Effect of Plasma Fibrinogen Levels on the Risk of Stroke in Patients with Type 2 Diabetes: A Systematic Review

Aims  In this systematic review, we assessed the literature on the association between fibrinogen levels and stroke in patients with type 2 diabetes (T2D). Methods  MEDLINE and Ovid searches of English reports were performed on the relation between fibrinogen, stroke, and T2D in humans. The search was completed on May 4, 2023. Studies were eligible when T2D patients ≥18 years had stroke confirmed by computed tomography or magnetic resonance imaging, plasma fibrinogen was measured, and a relation between fibrinogen and stroke in T2D patients was reported. Screening of reports and extraction of data were done independently by two authors, and study quality was assessed by predefined issues. Results  Five studies of different designs were included. Three studies reported on significantly increased fibrinogen levels in T2D patients with stroke compared with T2D patients without stroke. Two studies did not observe a significant association between fibrinogen levels and stroke risk. Conclusion  No consistent association was observed between fibrinogen levels and risk of stroke in T2D patients. Due to differences in study design, low sample size, and poorly defined study participants, larger and better-defined studies are needed to elucidate the role of fibrinogen as a stroke risk marker in T2D patients.


Introduction
Ischemic stroke (IS) is caused by a thrombus in a cerebral artery leading to reduced blood supply to the brain tissue.In IS, the clot can be formed in the brain itself (thrombotic stroke) or away from the brain and carried cause of death and the third leading cause of death and disability combined. 2n important risk factor for stroke is diabetes, and patients with diabetes are at more than twice the risk of getting an IS than nondiabetic people. 3In fact, diabetes increases the risk of a first stroke as well as stroke recurrence. 4Diabetes affects 10.5% of adults (20-79 years) worldwide and the majority are diagnosed with type 2 diabetes (T2D). 5The increased risk of stroke and cardiovascular diseases in general among T2D patients is not fully explained by the major risk factors (hypertension, smoking, and hypercholesterolemia). 6It may also be due to the procoagulant state seen in diabetes patients or dysfunction of the vascular homeostasis. 7The prevalence of T2D is increasing worldwide and is expected to rise even more in the future. 5][10] Understanding the underlying mechanisms may help in predicting, preventing, and treating stroke in this growing group of patients.
A possible mechanism via which diabetes can be associated with risk of stroke may involve fibrinogen, a central protein in the coagulation system, and an independent risk factor for stroke and recurrent stroke in the general population. 11,12Studies have demonstrated that levels of fibrinogen are increased in T2D. 6,13,14Understanding the relationship between stroke, diabetes, and fibrinogen is valuable in the prediction and treatment of stroke.Fibrinogen is a 340-kDa glycoprotein and is present in the circulation in a concentration of 2 to 5 g/L, but it can exceed 7 g/L during acute inflammation. 15When fibrinopeptides are cleaved off by thrombin, fibrin monomers are generated that polymerize into long fibers and ultimately into a fibrin clot. 16][19] Although several studies showed a relationship between increased concentrations of fibrinogen and stroke in the general population, 11,12 only a limited number of studies have focused on the association between fibrinogen and stroke in the growing population of patients with T2D.This systematic review summarizes the results of studies analyzing the association between fibrinogen levels and the risk of stroke (IS, HS, and TIA) in patients with T2D.

Methods
This systematic review was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. 20It was registered on PROSPERO (registration number: CRD42021286074), and no review protocol was conducted.
We excluded MEDLINE journals in the Ovid search, and there was no restriction on year of publication.The last search was completed on May 4, 2023.

Study Selection
Two researchers (E-M.B. and N.D.) independently screened records in two steps using Covidence systematic review software, Veritas Health Innovation, Melbourne, Australia (available at www.covidence.org).First, we identified records based on their title and abstract.Next, we assessed the selected reports through full-text screening.Reports were considered eligible when they met the following criteria: involving patients !18 years of age with T2D and IS, HS, or TIA.The diagnosis of stroke should be confirmed by computed tomography (CT) or magnetic resonance imaging (MRI), fibrinogen levels should be measured in plasma at any time, and the fibrinogen method must be reported.Finally, the study should report on the relationship between fibrinogen level and stroke in T2D patients.We excluded studies not conducted in humans, studies including only type 1 diabetes patients, reports published in languages other than English, reports with no full texts available, abstracts from congresses, letters, editorials, and study protocols.
The reference lists of included reports were assessed for relevant papers through PubMed on January 13, 2022.If fulfilling the inclusion criteria, the studies were included in the systematic review.Disagreements between the two researchers were solved by consensus or by the referee A-M.B.M. Remaining duplicates in the Ovid search were removed manually or removed by Covidence.

Data Extraction
Two researchers (E-M.B. and N.D.) collected data, independently of each other, to reduce the risk of bias.The following data were collected: first author, year of publication, country of study, type of study, sample size, type of imaging for stroke diagnosis, type of stroke, method used for fibrinogen measurement, and fibrinogen levels in T2D patients with stroke compared with T2D patients without stroke.

Quality Assessment
The scientific quality of studies included in a systematic review is of utmost importance for the overall conclusion.Since there are no standardized quality criteria, our expert author team decided upon seven issues of importance that should be evaluated for each of the studies, to clarify areas that might affect the results or the transparency of the studies.The issues were sample size considerations (to exclude type 2 errors), fibrinogen measured by a standard method (to compare fibrinogen concentrations between studies), and reporting of the following: duration of diabetes (affects the risk of stroke), characteristics of diabetes patients, medication, comorbidity (possible confounders), and statistical analysis (to judge the validity of study results).Based on these items, the studies were scored from 0 to 7, and all included studies were analyzed in the systematic review, irrespective of the quality score.

Study Selection
A total of 730 records were identified through the search.After the selection process, five studies were considered eligible for inclusion (►Fig.1).

Fibrinogen and Stroke Risk in Type 2 Diabetes Patients
A prospective study 21 measured fibrinogen at study enrolment and followed the patients for the occurrence of stroke for a median of 5 years.They demonstrated a significant increase in fibrinogen levels in T2D patients with stroke compared with T2D patients without stroke and found that fibrinogen levels were positively associated with an increased risk of stroke.Two case-control studies 22,23 showed a significant increase in fibrinogen levels in T2D patients in the nonacute phase at an undefined time point after stroke compared with T2D patients without stroke.Two studies did not observe any significant differences in fibrinogen between T2D patients with or without stroke in either a nested casecontrol design with >2 years follow-up 24 or a case-control design in which concentrations of fibrinogen were measured in the acute phase (within 24 h) after the stroke. 25The outcome stroke included TIA or HS in three of the studies, but substudies on stroke type in relation to fibrinogen were not performed.The results are summarized in ►Table 1.

Study Characteristics
Characteristics of individual studies are presented in ►Table 2. The five studies were published between 1990 and 2020, and they were performed in Asia, Australia/New Zealand, and Europe.The included studies were of different designs: one nested case-control study, one prospective study, and three case-control studies.
The sample size varied between the studies from 14 to 333 T2D patients with stroke.Three studies used CT as diagnostic tool and two used either CT or MRI.All studies reported on IS.Different methods were used to measure fibrinogen and only  two studies 21,24 used a standard method. 26The time point of fibrinogen measurement in relation to stroke onset differed between the studies.Blood samples were taken within 24 hours after development of stroke symptoms in one study, 25 in the nonacute phase after stroke in two studies, 22,23 and at the baseline examination in two prospective studies. 21,24

Patient Characteristics
The studies included more men than women, and the age range was comparable between studies.Detailed characteristics of patients with T2D and stroke/no stroke were described in two out of five studies (►Table 3).Laboratory data were comparable in these two studies, except for hemoglo-bin A1c (HbA1c) which was presented in two different units (mmol/mol and percent).In the study of Asakawa et al, 22 patients had T2D for a longer period, a larger percentage of patients were smokers, they had a lower BMI, less patients had hypertension, and more patients had nephropathy or neuropathy compared with the study of Hankey et al. 21The use of medication in the two studies was not comparable, and within the same study the intake of medication was different between the stroke and nonstroke groups. 21

Inclusion and Exclusion Criteria
Inclusion criteria in the studies were patients with T2D admitted to the hospital during 1 year 22 or within 24 hours  21 Australia, New Zealand, Finland Asakawa et al 2000 22

Japan
Ali et al 2020 23

India
Bots et al 2002 24 Finland, United Kingdom, The Netherlands Mistry et al 1990 25

India
Fibrinogen level T2D þ stroke T2D À stroke Risk estimate 3.72 (0.81) g/L a,c 3.58 (0.74) g/L a HR 1.The studies had different exclusion criteria, which are summarized in ►Table 4. Liver disease(s) was an exclusion criteria in four studies, and renal impairment was an exclu-sion criteria in three studies.Infections, HS, inflammatory diseases, history of myocardial infarction, and surgery within preceding 3 months were reasons for exclusion in two studies.Most of the exclusion criteria (14/24) came from the study of Ali et al. 23

Quality Assessment
Seven issues of importance in the quality assessment of the five studies are reported in ►Table 5 as covered (þ) or not covered (À) in the studies.Based on the quality issues Table 4 Exclusion criteria Hankey et al 21 Asakawa et al 22 Ali et al 23 Bots et al 24 Mistry et al 25 Hemorrhagic

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Table 5 Quality assessment Hankey et al 21 Asakawa et al 22 Ali et al 23 Bots et al 24 Mistry et al 25 Sample size considerations  defined by us, the overall quality of the included studies was low (three studies scored 0-1 out of 7).

Discussion
We identified five relevant articles by screening the literature.These articles fulfilled our inclusion criteria as studies that analyzed the association between fibrinogen and the risk of stroke in patients with T2D.One study observed higher baseline concentrations of fibrinogen in T2D patients who later developed stroke than in T2D patients who did not develop stroke.2][23] Two studies did not observe any significant differences in fibrinogen between T2D patients with or without stroke, either prospectively or in the acute phase after stroke. 24,25To our knowledge, this is the first systematic review investigating the association between fibrinogen and risk of stroke in patients with T2D.This was confirmed by screening the literature and by reviewing the registry for systematic review protocols, PROSPERO.The largest of the five studies and the one with the highest quality is the study by Hankey et al. 21To recruit a study population representing the general T2D population, the study had wide inclusion criteria, and T2D patients aged 50 to 75 years were included.Fibrinogen levels were measured in 9,795 T2D patients at enrolment in the study, and the patients were followed for the occurrence of stroke for a median of 5 years.They found that fibrinogen levels were associated with an increased risk of stroke (hazard ratio ¼ 1.14).This risk estimate is lower than the nonsignificant odds ratio of 1.9 in the nested case-control study by Bots et al. 24 This odds ratio was calculated from only 46 T2D patients including an unknown number of stroke cases in a study population characterized by only age and sex.In both prospective studies, fibrinogen levels were measured using a standard method.
Among the case-control studies, the study by Asakawa et al 22 is of highest quality, fulfilling all our criteria.They found significantly increased fibrinogen levels in T2D patients with stroke compared with T2D patients without stroke, results that were confirmed in the case-control study by Ali et al. 23 In both studies, blood samples were obtained in the nonacute phase at undefined time points after stroke.The case-control study by Mistry et al 25 reported on fibrinogen levels in the acute phase after stroke.Measurements of an acute phase reactant in the acute phase of stroke are not comparable with data from studies with measurement in the nonacute phase after stroke. 27None of the case-control studies used a standard method for fibrinogen measurements, 26 further complicating comparisons between the studies.
A general study limitation is the sample size.Four of the studies included less than 63 patients with stroke and T2D, [22][23][24][25] and sample size might explain the nonsignificant findings of fibrinogen in relation to stroke in the two studies with the fewest T2D patients. 24,25Only one study considered the size of the study population, 21 even though an appropri-ate sample size is needed to avoid a statistical risk of type 2 errors, also in subgroups of diabetes patients.Only three of the studies [21][22][23] reported on the statistical analysis, making it difficult to judge the validity of the results for fibrinogen as a risk factor for stroke in T2D patients.
Detailed information about the diabetes patients' characteristics was only given in two studies in which the duration time of T2D was very different. 21,22The remaining three studies [23][24][25] did not report on duration of diabetes even though increased risk of diabetes-related complications such as atherosclerosis, neuropathy, and nephropathy are well-known, long-term effects of T2D. 28The long-term disease risk associated with diabetes can be reduced by intensive diabetes control, but HbA1c was only reported in two studies. 21,220][31] The sex distribution within the group of stroke/no stroke was only given in two of the studies. 21,23his may affect the results since fibrinogen levels are higher in women, and the risk of stroke is more frequent in women with T2D than men with T2D. 3,32There were more smokers among T2D patients with and without stroke in the study of Asakawa et al 22 compared with the study by Hankey et al, 21 and three studies did not report on the distribution of smokers.The ethnicity of the study participants was not presented, however, it is noteworthy that two of the studies reporting a significant increase in fibrinogen levels in stroke patients with T2D were performed in Asia. 22,23Waist-hip ratio was only reported by Hankey et al, 21 even though the waist-hip ratio is suggested to be a better estimate of visceral fat than BMI. 33The abovementioned patient characteristics will most likely affect the association between fibrinogen and stroke in T2D patients.This important missing information in three studies [23][24][25] makes it difficult to compare the patient groups and the results from the five included studies.
5][36] However, AF was only reported by Hankey et al. 21Medication such as platelet inhibitors and lipid-lowering drugs are known to decrease fibrinogen levels, 31 and antithrombotic medication affects the risk of stroke. 37][24][25] Inclusion criteria differed in all studies and included different age groups, men only or both women and men, and time of stroke symptoms.Exclusion criteria also varied, making it difficult to compare the outcome.In some of the studies, exclusion criteria were infections, cancer, or previous cardiovascular events, all known to increase fibrinogen levels 16,38,39 and the risk of stroke, 30,40,41 and intake of antithrombotic medication, which decreases the risk of stroke. 37These criteria should preferably have been exclusion criteria in all the studies. 16 limitation in this review is that we might have missed studies in the literature search.However, by carefully selecting the search criteria, searching two databases, and searching the reference lists of included papers, we believe that we have covered a major part of available publications.
In conclusion, we identified five relevant studies of which three observed a significant positive association between fibrinogen levels and risk of stroke in patients with T2D.Due to differences in study design, low sample size, and missing information related to patient characteristics we were not able to clearly identify or reject fibrinogen as a predictor of stroke in T2D patients.Our results demonstrate that there is a clear need for more, larger, and better-defined studies to elucidate the role of fibrinogen as a marker of stroke risk in patients with T2D.If studies of high scientific quality can identify fibrinogen as an independent stroke risk factor in patients with T2D, future studies must address causality and underlying mechanisms, including effects of variants of the fibrinogen molecule.This new knowledge may be a valuable help in predicting, preventing, and treating stroke in the large population of T2D patients.

Fig. 1
Fig. 1 Preferred Reporting Items for Systematic Reviews and Meta-Analyses flowchart showing identification of included reports.CT, computed tomography; MRI, magnetic resonance imaging; T2D, type 2 diabetes.

Fibrinogen
and Stroke Risk in Type 2 Diabetes Daugaard et al.

Fibrinogen and Stroke Risk in Type 2 Diabetes
Daugaard et al. e75 Abbreviations: BMI, body mass index; HbA1c, hemoglobin A1c; HDL, high-density lipoprotein; T2D, type 2 diabetes.Note: Data are expressed as mean (standard deviation), median (interquartile range), or[range]."-"indicates that the value was not presented in the study.a mmol/mol b %

Fibrinogen and Stroke Risk in Type 2 Diabetes
Daugaard et al. after development of stroke symptoms.25Age was also an inclusion criteria with T2D patients aged 50 to 75 years,21 whereas the study of Bots et al24 included men aged 42, 48, 54, or 60 years (Finland), men aged 45 to 59 years (United Kingdom), and men and women !55 years (The Netherlands).Ali et al23 included patients based on purposive sampling technique from inpatient and outpatient departments.

Fibrinogen and Stroke Risk in Type 2 Diabetes
Daugaard et al. e77

Table 1
Fibrinogen and stroke risk in type 2 diabetes patients

Table 3
Characteristics of patients with type 2 diabetes with and without stroke